The Group A Streptococcus (GAS) has the ability to survive in many host tissues. It is important to have a better understanding of the mechanisms by which this pathogen can adapt and persist at the different sites within the host. The GAS cell utilizes many regulatory pathways in order to sense the host environment; one such pathway is the Mga virulence regulon. In this application, we hope to identify the role Mga plays as a virulence regulator as well as its role in cellular physiology through biochemical and genetic studies. The strain 5448-AN, whose genome sequence is available to us, will be used as a representative of the invasive and globally disseminated M1T1 GAS clone. In order to identify genes that are important for Mga regulation, we will use a Pmga-gusA reporter allele within the M1T1 5448- AN genome. This strain will be subjected to saturating transposon mutagenesis in order to create a mutant library using a newly developed mariner-based transposon (OSKAR), which has been optimized for efficient random mutagenesis within GAS. By identifying genes required for Mga regulation, we can begin to assemble the direct and indirect interactions of genes on the transcription of mga. We will also use the innovative genomic methodology of deep sequencing of transposon mutants (Tn-seq) in order to identify genetic interactions of the mga gene. This will provide us with a better understanding of how the Mga virulence regulator contributes to both pathogenesis and cellular physiology. This will be accomplished by using OSKAR, to create saturated mutant libraries in both wild type and mga 5448-AN backgrounds. We will determine the fitness of genes that are utilized for growth in rich medium (THY). In order to better characterize our findings, a selected number of genes shown to be involved in interacting with mga will be chosen for further analysis to identify their role in GAS virulence. The overarching goal of the work proposed in this fellowship is to define the full extent of the Mga regulatory network and how it links virulence and different aspects of cell physiology in the clinically relevant M1T1 GAS using global genetic analyses. The successful completion of these studies will help to resolve the complexity and interactions between regulatory networks important for GAS pathogenesis.